Description

THP-1 (DRB1*01:01/13:01) cells were differentiated into macrophages by stimulating with PMA (10 ng/ml) and IFN-gamma (200 activity units/ml). A total of 12 T-175 flasks were used to generate the macrophages from which approximately 0.3 grams cell pellet was obtained. The cell pellet was processed to extract cell membrane bound MHC-peptide complexes. The complexes were then ran through a tandem W632 and L243 affinity columns to purify MHC I and II complexes respectively. The eluted complexes were then acid boiled to release peptides from the MHC grooves. The peptides were then fractionated in RP-HPLC, dried up in speed vaccum, resuspended in 10% sequencing grade acetic acid spiked with iRT peptides, and ran on nanoLCMS (TTOF) system. The raw DDA data were denovo sequenced and peptides were identified using PEAKS software at 1% FDR. Before the cell pellet was processed, cells were tested for HLA-DRB1 expression by flow cytometry.

MHC II expression increases following PMA and interferon gamma stimulation

A table of some MHC I peptides and including a Biognosys iRT peptide is shown below.

A total of 857 class I peptides including 11 iRT peptides were detected of which most of them were 9-mers.

HLA-A02:01 motif

THP-1 W632 motif

A table of some MHC II peptides is shown below.

A total of 1404 class II peptides including 11 iRT peptides were detected of which most of them were 15-mers.

HLA-DRB1*01:01 motif

THP-1 L243 motif

Peptide sampling comparing the class I and class II derived peptides looked interesting.

For class I a protein was sampled one time for about 73% of the proteome whereas for class II a protein was sampled one time for only about 37% of the proteome (50% less than class I).